Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

Abstract

Canonical growth factors act indirectly via receptor-mediated signal transduction pathways. Here, we report on an autonomous pathway in which a growth factor is internalized, has its localization regulated by phosphorylation, and ultimately uses intrinsic catalytic activity to effect epigenetic change. Angiogenin (ANG), a secreted vertebrate ribonuclease, is known to promote cell proliferation, leading to neovascularization as well as neuroprotection in mammals. Upon entering cells, ANG encounters the cytosolic ribonuclease inhibitor protein, which binds with femtomolar affinity. We find that protein kinase C and cyclin-dependent kinase phosphorylate ANG, enabling ANG to evade its inhibitor and enter the nucleus. After migrating to the nucleolus, ANG cleaves promoter-associated RNA, which prevents the recruitment of the nucleolar remodeling complex to the ribosomal DNA promoter. The ensuing derepression of rDNA transcription promotes cell proliferation. The biochemical basis for this unprecedented mechanism of signal transduction suggests new modalities for the treatment of cancers and neurological disorders.

ANG degrades pRNA in cellulo. (A) Graph of qRT-PCR data indicating that ANG (1 μg/ml) reduces the level of pRNA in HeLa cells by 50%, which is the same level achieved with LNA-antisense knockdown done as described previously (). Values represent the mean ± SD (n = 3, biological replicates). (B) Graphs showing that ANG variants have differential effects on pRNA levels in vitro (left) and in cellulo (right). S28N ANG, which is an active enzyme in vitro but defective in nuclear localization (), leads to no change in pRNA level in cellulo. C39W ANG, which is unstable, reduces pRNA level by only 25% in cellulo. H114R ANG, which has a deleterious active-site substitution, leads to no change in pRNA levels in vitro or in cellulo. Q117G ANG, which has an advantageous active-site substitution, leads to highly reduced pRNA levels in vitro and in cellulo. Values represent the mean ± SD (n = 3, in vitro: technical replicates, in cellulo: biological replicates). Paired Student's t-test: differences were considered significant at *P < 0.05. (C) An RNA co-immunoprecipitation with FLAG–H114R ANG (1 μg/ml) demonstrates a direct interaction between ANG and pRNA in cellulo. EtBr-stained agarose gel of PCR products that were amplified from the pRNA region corresponding to primer 1 (P1) and primer 2 (P2). Only the P2-derived pRNA region was detected in the IP samples, indicating that this region was protected by FLAG–H114R ANG. The P1-derived pRNA region as well as other cellular RNAs were vulnerable to degradation by RNase A. The PCR product of P2 was sequenced and its identity was confirmed as the conserved stem-loop structure of pRNA.

Cleavage of pRNA by ANG Promotes Dissociation of TIP5 in Cellulo. (A) Immunofluorescence images of nucleolar TIP5 (green) in HeLa cells indicating that ANG (1 μg/ml) limits the accumulation of TIP5 in the nucleolus. Blue: Hoechst 33342. Scale bar: 20 μm. (B) Autoradiograms of gel-shift assays indicating that ANG and the hyperactive Q117G variant degrade the pRNA within a TAM·pRNA complex in vitro; the inactive H114R variant does not. (C) Graphs of chromatin immunoprecipitation at the rDNA promoter revealing that treatment with ANG and the Q117G variant enrich the occupancy of H3K4me3 but decrease the occupancy of H3K9me3, which is a marker of repressive transcription. The H114R variant does not change H3K4me3 or H3K9me3 levels. Values represent the mean ± SD (n = 3, biological replicates). Paired Student's t-test: differences were considered significant at *P < 0.05.

ANG is phosphorylated by PKC and CDK. (A) Structure of the human RI·ANG complex (PDB:1A4Y) (). Putative phosphorylation sites in ANG (blue ribbon) are labeled and depicted in ball-and-stick. The Coulombic surface of RI (grey ribbon) is depicted with red = negative and blue = positive. Inset: Close contact between Ser87 of ANG and two tryptophan residues of RI. (B) Autoradiogram of a polyacrylamide gel demonstrating that ANG is phosphorylated upon incubation with a HeLa cell lysate and [γ-32P]ATP. Replacing Thr36/Ser37 or Ser87 with an alanine residue decreases phosphorylation. An immunoblot of the same gel shows consistent loading of ANG and its variants. (C) Immunoblots showing that FLAG–ANG (1 μg/ml) taken up by HeLa cells and isolated by immunoprecipitation (IP) with an anti-FLAG antibody (α-FLAG) is recognized by an anti-phosphoserine antibody (α-P-serine). Recognition is eliminated upon treatment with lambda protein phosphatase (LPP). IP of the S28N variant was reduced, and that of the T36A/S37A and S87A variants was reduced even more. (D) Immunoblots showing that small-molecule inhibitors of CDK or PKC reduce the phosphorylation of FLAG–ANG by HeLa cells. For kinase-inhibitor treatment, cells were pre-incubated with either bisindolylmaleimide (4 μM) or roscovitine (14 μM) for 30 min prior to the treatment with ANG. Representative immunoblots are shown in panels C and D, and independent biological replicates are shown in Supplementary Figure S5.

Scheme of the cellular action of ANG. Angiogenin binds to syndecan-4 on the cell surface and is internalized by endocytosis. A fraction translocates to the cytosol, where ANG is phosphorylated by PKC and CDK. Phosphorylation endows ANG with the ability to evade the ribonuclease inhibitor protein. Phosphorylated ANG translocates into the nucleus and accumulates in the nucleolus. There, ANG digests pRNA, leading to the dissociation of TIP5 from the rDNA promoter. The ensuing rDNA transcription fuels the proliferation of endothelial cells (neovascularization) and tumor cells (cancer progression).